Breeding Science 66: 148–159 (2016) doi:10.1270/jsbbs.66.148

Review

Genomics of and other

Toshiya Yamamoto* and Shingo Terakami

NARO Institute of Fruit Science, 2-1 Fujimoto, Tsukuba, Ibaraki 305-8605, Japan

The family Rosaceae includes many economically important fruit trees, such as pear, , , , , , , , and . Over the past few years, whole-genome sequences have been released for Chinese pear, European pear, apple, peach, Japanese apricot, and . These sequences help us to conduct functional and comparative genomics studies and to develop new cultivars with desirable traits by marker-assisted selection in breeding programs. These genomics resources also allow identification of evolutionary relationships in Rosaceae, development of genome-wide SNP and SSR markers, and construction of reference genetic linkage maps, which are available through the Genome Database for the Rosaceae web­ site. Here, we review the recent advances in genomics studies and their practical applications for Rosaceae fruit trees, particularly pear, apple, peach, and cherry.

Key Words: apple, co-linearity, genome sequence, peach, pear, reference map.

Introduction al. 1996). Loquat ( japonica (Thunb.) Lindl.) is also an important fruit tree that belongs to the tribe Pyreae The family Rosaceae consists of about 2500 species from along with and . Several (“stone fruit”) 90 genera and includes diverse , which are primarily species are also important fruit trees and include native to temperate regions (Hummer and Janick 2009). and nectarines (P. persica (L.) Batsch), (P. domestica This family was traditionally classified into several subfam­ L., P. salicina Lindl.), (P. armeniaca L., P. mume ilies: , , , , Siebold et Zucc.), and (P. avium L., P. cerasus L.), and others (Hummer and Janick 2009). In 2007, three sub­ which belong to the subfamily Amygdaloideae, tribe families were suggested: , Rosoideae and Amygdaleae. Annual world fruit production (in million Amygdaloideae (Potter et al. 2007); the latter subfamily tons) is as follows: peaches and nectarines, 21.6; plums, includes the former Amygdaloideae, Maloideae and 11.5; apricots, 4.1; and cherries 2.3 (FAOSTAT 2013). In Spiraeoideae. Many economically important crops produc­ Japan, domestic fruit production (in thousand tons) is as ing edible (e.g., apple, apricot, cherry, loquat, peach, follows: apples, 742; pears, 294; peaches, 125; apricots, pear, plum, quince, raspberry, and strawberry), nuts (e.g., al­ 124; plums, 21; and cherries, 18 (FAOSTAT 2013). mond), and ornamentals (e.g., ) belong to the Rosaceae. Basic chromosome number x = 7, 8, 9, 15 or 17 was ob­ The most economically important members of the served for Rosaceae members (Dirlewanger et al. 2009b, Rosaceae are apples ( × domestica Borkh.) and pears Evans and Campbell 2002, Potter et al. 2007). The subfami­ (Pyrus spp.), both of which belong to the subfamily ly Rosoideae, which contains rose, raspberry, and straw­ Amygdaloideae, tribe Pyreae. Annual world fruit production , has x = 7. The tribe Amygdaleae of subfamily of apples exceeds 80 million tons (FAOSTAT 2013), making Amygdaloideae, known for , apricot, cherry, peach, them the third most important fruit (after citrus and banana). and plum, has x = 8. The former subfamily Spiraeoideae Pears are the second most important fruit species in Rosaceae, (tribe Spiraeeae of subfamily Amygdaloideae,) has x = 9. with world production of approximately 25.2 million tons Basic chromosome number x = 17 is observed for the tribe (FAOSTAT 2013). Four important Pyrus species are com­ Pyreae of subfamily Amygdaloideae, which contains apple, mercially grown for edible fruit: Japanese pear (P. pyrifolia loquat, pear, and quince. Challice (1974, 1981) suggested Nakai), European pear (P. communis L.), and Chinese pears that the Pyreae was generated by an allopolyploidization (P. bretschneideri Rehd. and P. ussuriensis Maxim.) (Bell et event between “Amygdaleae” (x = 8) and “Spiraeeae” (x = 9). Recent molecular genetic studies contradicted the Communicated by M. Omura allopolyploidization and supported the autopolyploid origin Received September 10, 2015. Accepted January 12, 2016. of hybridization between closely related members of *Corresponding author (e-mail: [email protected]) Spiraeeae (Evans and Campbell 2002). Velasco et al. (2010)

148 Breeding Science Genomics of pear and other Rosaceae fruit trees Vol. 66 No. 1 BS showed that a relatively recent (ca. 50 million years ago) and covers a total of 577.3 Mb (96.2% of the estimated genome-wide duplication resulted in the transition from genome size, 600 Mb). A total of 43,419 putative genes nine ancestral chromosomes to 17 chromosomes in apple, were predicted, of which 1219 are unique to European pear based on whole-genome sequencing analysis. and are not found in other dicots genomes sequenced. In recent years, international collaborative studies by the Analysis of the expansin gene family and other cell wall- Rosaceae research community have hastened progress in related genes showed their involvement in fruit softening developing genetic and genomic resources for representa­ in both European pear and apple. It is expected that pear tive crops such as apple (M. × domestica), peach (P. persica), genome sequences of Chinese and European pears will be and strawberry ( spp.) (Shulaev et al. 2008); this assigned to 17 pseudo-chromosomes, which will greatly strategy was based on a consensus that there are multiple help us to conduct genetics and genomics studies in pears. Rosaceae model species (Dirlewanger et al. 2009b). These An international consortium has published a draft ge­ resources, including expressed sequence tags (ESTs), bac­ nome sequence of the domesticated apple ‘Golden terial artificial chromosome (BAC) libraries, physical and Delicious’, a common founder cultivar in many breeding genetic maps, and molecular markers and bioinformatics programs (Velasco et al. 2010). The genome assembly of tools, are available through the Genome Database for the ‘Golden Delicious’ consists of 122,146 contigs spanning a Rosaceae (GDR; http://www.rosaceae.org). The availability total of 603.9 Mb (81.3% of the estimated genome, of this database has rendered various rosaceous crops highly 742.3 Mb). Seventeen pseudo-chromosomes (GDR, Malus amenable to functional and comparative genomics studies. × domestica Genome v1.0p) were obtained from these con­ Here we review recent progress in genomics studies on tigs. A total of 57,386 putative protein-coding genes were Rosaceae fruit trees such as apple, pear, peach, and cherry, predicted. The MADS-box gene family involved in flower and we discuss the newly accumulated knowledge and re­ and fruit development is expanded in apple to 15 members. sources for comparative genomics studies on this family. The other gene families related with transport and assimila­ tion of are also expanded, and are involved in Genome sequences of Rosaceae fruit crops Rosaceae-specific metabolism. A high-quality draft reference genome sequence, Peach Whole-genome sequences have been reported for Chinese v1.0, of the doubled haploid genotype of the peach cultivar pear (Wu et al. 2013), European pear (Chagné et al. 2014), ‘Lovell’ has been reported (Verde et al. 2013). Since apple (Velasco et al. 2010), peach (Verde et al. 2013), Japa­ ‘Lovell’ is completely homozygous, its genome assembly nese apricot (Zhang et al. 2012), wild strawberry (Shulaev has facilitated obtaining a reliable and unbiased reference et al. 2011) and cultivated strawberry (Hirakawa et al. genome. Using 827 markers from an updated Prunus ref­ 2014) (Table 1). The draft genome of the Chinese pear erence map (Howad et al. 2005), Verde et al. (2013) or­ ‘Dangshansuli’ (P. bretschneideri) is now available (Wu et ganized 215.9 Mb of the Peach v1.0 genome into eight al. 2013). A total of 2103 scaffolds span 512.0 Mb (97.1% pseudomolecules covering 81.5% of the estimated genome of the estimated genome size, 527 Mb) and are not anchored (265 Mb). A total of 27,852 protein-coding genes were pre­ to the 17 chromosomes. The Chinese pear genome assembly dicted. Furthermore, comparative analyses showed that the contains 42,812 protein-coding genes, and about 28.5% of ancestral triplicated blocks in peach are detected, and that them encode multiple isoforms. The identified repetitive se­ putative paleoancestor regions are detectable. quences (271.9 Mb in total) account for 53.1% of the ge­ The genome of Japanese apricot or mei (P. mume) was nome. The difference in size between the pear and apple one of the first genomes to be sequenced in the subgenus genomes is mainly due to the presence of repetitive se­ Prunus of the Prunus (Zhang et al. 2012). Japanese quences (predominantly transposable elements), whereas apricot was domesticated in China more than 3000 years genic regions and protein-coding genes are similar in both ago as an ornamental plant and fruit tree. A 237-Mb genome species. A draft genome assembly of European pear assembly was generated from 29,989 scaffolds, 84.6% of ‘Bartlett’ (Chagné et al. 2014) contains 142,083 scaffolds which were further anchored to eight chromosomes in a

Table 1. Details of whole-genome sequencing in Rosaceae crops Pyrus bretschneideri Malus × domestica Prunus persica Common name Chinese pear European pear apple peach Japanese apricot woodland strawberry Cultivar name Dangshansuli Bartlett Golden Delicious Lovell BJFU1210120008 Hawaii 4 (PI551572) No. of contigs 25,312 182,196 122,146 – 45,592 – No. of scaffolds 2103 142,083 – 391 29,989 3263 Genome assembly size (Mb) 512.0 577.3 603.9 215.9 237 209.8 Coverage (%) 97.1 96.2 81.3 81.5 84.6 95 Estimated genome size (Mb) 527 600 742.3 265 280 240 No. of putative genes 42,812 43,419 57,386 27,852 31,390 34,809 No. of pseudo-chromosomes – – 17 8 8 7 Reference Wu et al. 2013 Chagné et al. 2014 Velasco et al. 2010 Verde et al. 2013 Zhang et al. 2012 Shulaev et al. 2011

149 Breeding Science BS Vol. 66 No. 1 Yamamoto and Terakami genetic map constructed by restriction-site-associated DNA 2007, Sosinski et al. 2000, Testolin et al. 2000, Yamamoto sequencing (RADseq); 31,390 protein-coding genes were et al. 2002d, 2003, 2005), cherries (Cantini et al. 2001, annotated and integrated using ab initio gene prediction Downey and Iezzoni 2000, Joobeur et al. 2000, Struss et al. methods. By comparison of the P. mume genome with the 2002), and apricot (Lopes et al. 2002). available data, nine ancestral chromosomes of the Rosaceae family were reconstructed (Zhang et al. 2012). SNP markers Strawberry is one of the most important Rosaceae crops, Although at present SSR markers seem to be the best and genomes were sequenced for wild woodland strawberry choice for genetics and genomics studies, marker systems (Shulaev et al. 2011) and cultivated octoploid strawberry with even higher throughput, such as single-nucleotide (Hirakawa et al. 2014). The woodland strawberry F. vesca polymorphisms (SNPs), have been developed based on (2n = 2x = 14), a diminutive herbaceous perennial, has a whole-genome sequencing data. Using NGS technology, small genome (240 Mb) that shares substantial sequence Montanari et al. (2013) have developed 1096 SNPs from identity with the genomes of the cultivated strawberry three European pear cultivars. A total of 857 polymorphic (F. × ananassa) and other economically important rosa­ SNP markers were validated and mapped using a segregat­ ceous plants. A total of 209.8 Mb (>95%) of the genome ing population of European pear ‘Old Home’ × ‘Louise Bon sequence were included in 272 representative scaffolds out Jersey’ and interspecific breeding families derived from of 3262 scaffolds, which were anchored to seven pseudo- Asian (P. pyrifolia and P. bretschneideri) and European pear chromosomes in the genetic linkage map. Gene prediction pedigrees. Japanese pear ‘Housui’ (syn. ‘Hosui’) has also modeling identified 34,809 putative protein-coding genes. been used for EST sequencing of 185 Mb and genome se­ Macrosyntenic relationships between Fragaria (x = 7) and quencing of 529 Mb (Terakami et al. 2014). Using the Prunus (x = 8) predict a hypothetical ancestral Rosaceae GoldenGate assay, Terakami et al. (2014) evaluated 1536 genome that had nine chromosomes. Furthermore, the SNPs detected in EST and genome sequences of ‘Housui’, whole genome sequences of peach, apple and strawberry and mapped 609 SNPs on its linkage map. Using RADseq, were analyzed and compared by using 1399 orthologous re­ Wu et al. (2014) have genotyped Chinese pear SNPs by gions between the three genomes, suggesting the ancestral NGS and mapped 3143 SNPs on a linkage map. genome (x = 9) to the extant Fragaria, Prunus and Malus The 8K apple Infinium SNP chip has been developed by genomes (Illa et al. 2011, Jung et al. 2012). the USA-based international research program RosBREED (Chagné et al. 2012). To discover genome-wide SNPs, 27 Genome-wide molecular markers apple cultivars were chosen to represent worldwide breeding germplasms and were re-sequenced at low coverage by NGS SSR markers technology. Of 2,113,120 SNPs detected, 7867 were selected Simple sequence repeat (SSR) markers, or microsatel­ for the apple 8K SNP array; after evaluation in segregating lites, provide a reliable method for evaluation of genetic families and a germplasm collection, 5554 were found to diversity and construction of genetic maps because of their be polymorphic (Chagné et al. 2012). Despite this progress, co-dominant inheritance and the allelic abundance (Weber the number of robust and evenly distributed SNP markers and May 1989). More than 1000 SSR markers have been in the 8K array was not sufficient. Recently, a 20K SNP developed in Japanese and European pears from genome array has been developed by the European research program sequences (Fernández-Fernández et al. 2006, Inoue et al. FruitBreedomics, which focuses on bridging the gap be­ 2007, Sawamura et al. 2004, Yamamoto et al. 2002a, 2002b, tween breeding and genomics (Bianco et al. 2014). This SNP 2002c ), ESTs (Nishitani et al. 2009, Zhang et al. 2014), and array has been developed to enable high-precision genome- next-generation sequencing (NGS) data (Yamamoto et al. wide association analyses and pedigree-based analysis be­ 2013). Recently, a large number of SSR markers have been cause of rapid decay of linkage disequilibrium. The SNPs developed from the whole-genome sequence of Chinese included in this array were predicted from re-sequencing pear ‘Dangshansuli’ (Chen et al. 2015). SSR markers devel­ data derived from the genome sequences of 13 apple culti­ oped in pear have been often used as anchor loci for refer­ vars and one accession of crab apple (M. micromalus). ence genetic linkage maps of pear (Chen et al. 2015, Using NGS technology, the International Peach SNP Yamamoto et al. 2007). Consortium has re-sequenced the whole genomes of 56 In apple, hundreds of SSR markers have been developed peach breeding accessions (Verde et al. 2012, 2013) and (Celton et al. 2009, Gianfranceschi et al. 1998, Guilford developed a 9K SNP array (Verde et al. 2012). Using the et al. 1997, Liebhard et al. 2002, 2003, Moriya et al. 2012, GoldenGate assay, Martínez-García et al. (2013) have eval­ Silfverberg-Dilworth et al. 2006, van Dyk et al. 2010) and uated a set of 1536 SNPs of peach (P. persica) developed used to construct high-quality genetic linkage maps with from the whole-genome sequences of three cultivars. The high marker density. Among Prunus spp., a large number of RosBREED Consortium has also developed a 6K SNP array SSR markers have been developed for peach and almond for diploid sweet cherry (P. avium) and allotetraploid sour (Aranzana et al. 2002, 2003, Cipriani et al. 1999, cherry (P. cerasus) (Peace et al. 2012). Dirlewanger et al. 2002, Howad et al. 2005, Nishitani et al.

150 Breeding Science Genomics of pear and other Rosaceae fruit trees Vol. 66 No. 1 BS Reference genetic linkage maps Dilworth et al. 2006). Recently, the 8K Infinium SNP chip described above was used to construct a high-density genet­ High-density reference genetic linkage maps constructed ic linkage map in apple (Chagné et al. 2012). In the with genome-wide molecular markers are important for FruitBreedomics project, 21 full sib families were SNP- many genetic and breeding applications in Rosaceae fruit genotyped, resulting in the genetic mapping of approxi­ trees including marker-assisted selection (MAS), mapping mately 15,800 SNP markers (Bianco et al. 2014). of quantitative trait loci (QTLs), identifying DNA markers for fingerprinting, and map-based gene cloning. Because Prunus reference maps good, comprehensive books and reviews have been pro­ The framework Prunus mapping population for construc­ duced that describes mendelian traits and QTLs in Rosaceae tion of the reference map was an F2 population (referred to fruit trees (Dirlewanger et al. 2009a, Korban and Tartarini as the T × E population) produced by crossing almond 2009, Salazar et al. 2014), it would be impractical to repeat (Prunus dulcis) ‘Texas’ × peach (P. persica) ‘Earlygold’ and that information. Instead we describe high-density reference selfing a single F1 plant (MB 1-73) (Joobeur et al. 1998). genetic linkage maps in pear, apple and Prunus. The T × E map contained 562 marker loci (Dirlewanger et al. 2004a). Howad et al. (2005) established a Prunus refer­ Pear reference maps ence map using a set of six F2 plants, one F1 hybrid, and one Among Pyrus spp., integrated high-density genetic link­ parent of the F1 hybrid, which could jointly define 65 possi­ age maps are available for the European pear cultivars ble different genotypes by the markers mapped on the T × E ‘Bartlett’ and ‘La France’ and the Japanese pear cultivar map. Howad et al. (2005) identified and mapped 264 SSR ‘Housui’; these maps are based on SSRs from pear, apple, and markers from 401 different SSR primer pairs. Recently, Prunus, amplified fragment length polymorphisms (AFLPs), Verde et al. (2013) have aligned the eight main scaffolds isozymes, and phenotypic traits (Terakami et al. 2009, (pseudo-chromosomes) against the updated version of the Yamamoto et al. 2002c, 2004a, 2007). The linkage maps of Prunus reference map constructed by Howad et al. (2005). ‘Bartlett’, ‘La France’, and ‘Housui’ consisted of 447, 414, A consensus cherry genetic linkage map has been devel­ and 335 marker loci, respectively, and covered 17 linkage oped using 94 individuals from an interspecific cross, groups (LGs), which matched the basic chromosome num­ ‘Napoleon’ (P. avium) × P. nipponica accession F1292; this ber of pear (x = 17). Recently, Terakami et al. (2014) estab­ map consisted of 174 loci, including 160 SSR loci and 6 lished a SNP assay to evaluate 1536 SNPs detected in the gene-specific markers, and covered 680 cM (Clarke et al. EST and genome sequences of ‘Housui’, and mapped 609 2009). Cabrera et al. (2012) developed a sweet cherry SNPs on a linkage map of ‘Housui’. After all available SNP (P. avium) reference linkage map using Rosaceae Con­ and SSR markers were integrated, the latest version of up­ served Orthologous Set (RosCOS) markers and SSR mark­ dated reference genetic linkage map of ‘Housui’ was recon­ ers. RosCOS markers were identified from 3818 rosaceous structed (Fig. 1), which consists of 1033 loci, including 609 unigenes comprised of two or more ESTs corresponding to SNPs from transcriptome and genome analyses, 61 SNPs single-copy genes in Arabidopsis (Cabrera et al. 2009, from potential intron polymorphism markers (Terakami et 2012). Of the 627 RosCOS markers, 81 SNPs representing al. 2013), 202 pear SSRs, 141 apple SSRs, and 20 other 68 genome-wide RosCOS were mapped in four F1 popula­ markers. Montanari et al. (2013) evaluated a set of 1096 tions and placed on the consensus sweet cherry linkage map European pear SNPs and 7692 apple SNPs, and mapped 857 that included previously reported SSRs, indel, and S-RNase and 1031 SNPs, respectively, on pear genetic maps. On the markers and spanned 779.4 cM. Klagges et al. (2013) con­ basis of whole-genome sequencing of P. bretschneideri, structed SNP-based high-density genetic maps of sweet Chen et al. (2015) constructed a consensus genetic map cherry using intraspecific progenies from crosses between consisting of 734 SSR loci derived from 1341 newly de­ parental lines ‘Black Tartarian’ × ‘Kordia’ (BT × K) and signed SSRs. Using RADseq, Wu et al. (2014) mapped ‘Regina’ × ‘Lapins’ (R × L). Of 5696 SNP markers tested, 3143 SNPs on linkage maps of Chinese pear. 723 and 687 were mapped onto eight LGs in BT × K and R × L, respectively. The obtained maps spanned 752.9 and Apple reference maps 639.9 cM, with an average distance between markers of 1.1 Several apple reference genetic linkage maps have been and 0.9 cM, respectively. Very recently, genotyping-by- published. The first RFLP-based reference maps for ‘Prima’ sequencing (GBS), a new methodology based on high- and ‘Fiesta’ were constructed using 152 F1 individuals and throughput sequencing, was applied for genome mapping in the two maps were aligned using 67 multi-allelic markers sweet cherry (Guajardo et al. 2015). (Maliepaard et al. 1998). SSR-based integrated genetic link­ age maps for ‘Fiesta’ and ‘Discovery’ were constructed Marker-assisted selection in Japanese pear using 840 molecular markers including 129 SSRs (Liebhard et al. 2002, 2003). A new set of 148 apple microsatellite MAS can accelerate selection and reduce the progeny size markers has been developed and mapped on the reference and the cost of raising individuals to maturity in the field, linkage maps of ‘Fiesta’ and ‘Discovery’ (Silfverberg- especially in fruit trees (Luby and Shaw 2001). In Japanese

151 Breeding Science BS Vol. 66 No. 1 Yamamoto and Terakami

Fig. 1. The latest version of integrated reference genetic linkage map of Japanese pear ‘Housui’ based on SNP and SSR markers. A total of 81 SSR loci including 67 from pear ESTs or 454 genome sequencing analysis and 14 from apple, which were included in the ‘Housui’ map of Yamamoto et al. (2013), were added to the recently published SNP-based map (Terakami et al. 2014). Linkage groups are designated as Ho1 to Ho17, HoX1 and HoX2. The number to the left of each marker indicates genetic distance (cM). SSR markers (green, underlined) were developed from pear. SSR markers (red, italicized) were developed from apple. SNP markers developed by transcriptome analysis are denoted by JPsnpHou and SNP markers developed from potential intron polymorphism markers are denoted by TsuSNP. Distorted segregation is indicated by a signifi­ cant P value of the χ2 test: *P = 0.05, **P = 0.01, ***P = 0.005.

152 Breeding Science Genomics of pear and other Rosaceae fruit trees Vol. 66 No. 1 BS

Fig. 1. (continued)

153 Breeding Science BS Vol. 66 No. 1 Yamamoto and Terakami ready analyzed by genome mapping, QTL analysis, or both, the positions of responsible genes (loci) were identified in genetic linkage maps and tightly linked molecular markers were identified; these data are deposited in the public data­ base of the Applied Crop Genomics Research Center (http:// www.naro.affrc.go.jp/genome/index.html). DNA markers have been identified that are associated with genes for resistance to scab disease caused by Venturia nashicola (Gonai et al. 2012, Iketani et al. 2001, Terakami et al. 2006) and for resistance (or susceptibility) to black spot disease caused by a Japanese pear pathotype of Alternaria alternata (Banno et al. 1999, Iketani et al. 2001, Terakami et al. 2007). Self-incompatibility in Japanese pear is controlled by a single multi-allelic S-locus, and S-genotype identifica­ tion is important for breeding and selection of pollen donors for fruit production. Several molecular assays for rapid and reliable S-genotype determination have been established, such as polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) analysis (Ishimizu et al. 1999) and allele-specific PCR amplification (Nashima et al. sm 2015). The S4 allele of the self-compatible cultivar ‘Osa- Nijisseiki’ (a mutant of the self-incompatible cultivar ‘Nijisseiki’) has been identified and found to lack a 236-kbp genomic region that includes the S4-RNase coding region (Okada et al. 2008). Molecular markers associated with the following fruit-related traits were also revealed: fruit storage potential controlled by ethylene production (the 1- aminocyclopropane-1-carboxylate [ACC] synthase gene; Itai Fig. 1. (continued) et al. 2003), fruit skin color (Inoue et al. 2006, Yamamoto et al. 2014), and harvest time (Yamamoto et al. 2014). These markers can be used for MAS in Japanese pear breed­ pear, several molecular markers associated with genes of in­ ing programs. terest traits have been identified and used for MAS in practi­ cal breeding programs of the National Agriculture and Food Synteny in Rosaceae fruit trees Research Organization (NARO) Institute of Fruit Tree Sci­ ence, Japan (Table 2). Since several characteristics were al­ It is expected that comparative genomics in Rosaceae fruit

Table 2. Molecular markers associated with genes of interest in Japanese pear and their positions in genetic linkage maps Linkage Associsted molecular F-primer sequences R-primer sequences Characteristics Gene symbol Gene sources References group nos. markers (Accession nos.) (5′-3′) (5′-3′)a Scab resistance to Vnk Kinchaku 1 TsuENH184 (AB621908) cctccctcagtacccatcaa GTTTCTTtgaactccttcactcaccttcc Gonai et al. 2012, V. nashicola Terakami et al. 2006 TsuENH101 (AB621905) tgcctaatggaagggtccta GTTTCTTcaaggaagagaagaccgacg TsuENH157 (AB621907) tagcagcagctctcctccac GTTTCTTgtcagcacccctctgatgtt Black spot A Osa Nijisseiki 11 CMNB41/2350 gacagcgtccta Banno et al. 1999 susceptibility Ani Osa Nijisseiki 11 CH04h02 ggaagctgcatgatgagacc ctcaaggatttcatgcccac Terakami et al. 2007 CH03d02 aaactttcactttcacccacg GTTTCTTactacatttttagatttgtgcgtc Ana Nansui 11 CH04h02 ggaagctgcatgatgagacc ctcaaggatttcatgcccac Terakami et al. 2007 CH03d02 aaactttcactttcacccacg GTTTCTTactacatttttagatttgtgcgtc Self-incompatibility S Japanese pear 17 S-RNase tttacgcagcaatatcag acrttcggccaaataatt Ishimizu et al. 1999, Nashima et al. 2015 sm Self-compatibility S4 Osa Nijisseiki 17 SM tcgtcttagggatttccaatgc gccttaagggttcattgggc Okada et al. 2008 Fruit skin color Niitaka 8 OPH-19-425 ctgaccagcc Inoue et al. 2006 FruC Akiakari 8 Mdo.chr8.10 tgcagccctcaaacttttct caacccaactccagcaattt Yamamoto et al. 2014 CH04g12 caccgatggtgtcaacttgt caacaaaatgtgatcgccac Fruit storage PpACS2 Japanese pear 15 ACC synthase gtcacagaatcaacgattga agtagaacgcgaaaacaaat Itai et al. 2003 Harvest time HarT-1 (QTL) Taihaku 3 BGA35 (AB219799) agagggagaaaggcgatt GTTTCTTgcttcatcaccgtctgct Yamamoto et al. 2014 HarT-2 (QTL) Taihaku 15 PPACS2 ggtatctttgtccggcaatc gctctcaaggctttcttctctc Yamamoto et al. 2014 a GTTTCTT: pig tail sequence for DNA sequencer analysis.

154 Breeding Science Genomics of pear and other Rosaceae fruit trees Vol. 66 No. 1 BS trees will be able to integrate conserved candidate genes, proportion of SSRs showing polymorphism was also high molecular markers associated with interest traits, and QTLs, (63.9%) (Mnejja et al. 2010). In contrast, only 16.3% of the in order to verify how the genetic and molecular factors Prunus SSRs were transferable across species of other control traits like fruit quality and texture across species and Rosaceae genera such as apple, pear, and strawberry (Mnejja genera. Therefore, synteny or comparative genome mapping et al. 2010). is an important approach, which determines the homologous SSR markers developed for various Prunus species have genes of related species, as well as the co-linearity (conser­ been intensively used to compare Prunus linkage maps vation of the gene order) among conserved genomic regions. (Dirlewanger et al. 2004b). Detailed map comparisons were performed using common SSR markers between the refer­ Co-linearity between Pyrus and Malus ence genetic linkage map T × E (Joobeur et al. 2000) and Yamamoto et al. (2001) applied apple SSR markers inter­ the maps of P. armeniaca (Lambert et al. 2004), P. davidiana generically for the characterization of several pear species (Foulongne et al. 2003), and P. cerasifera (Dirlewanger et (P. pyrifolia, P. bretschneideri, P. ussuriensis, P. communis, al. 2004a). The distribution and order of SSR markers in all and P. calleryana). Nucleotide repeats were detected in the Prunus species show complete synteny except for a recipro­ amplified fragments of pear and apple by both sequencing cal translocation between LGs 6 and 8 detected in peach and and Southern blot analyses, and the differences in fragment almond (Dirlewanger et al. 2004b, Jáuregui et al. 2001). sizes between pear and apple were due mainly to the differ­ The SNP-based sweet cherry maps displayed high synteny ences in the number of such repeats. The SSR markers are and co-linearity of all eight LGs with the Prunus reference applicable across genera in the tribe Pyreae, subtribe Pyrinae, map and with the peach genome v1.0 (Klagges et al. 2013). which includes apple, pear, quince (Cydonia oblonga Mill.), and loquat (Liebhard et al. 2002, Soriano et al. 2005, Synteny between Pyrus (Malus) and Prunus Yamamoto et al. 2001, 2004a, 2004b). When pear genetic Transferability of SSR markers is very low between linkage maps (‘Bartlett’ and ‘La France’) were compared tribes, as shown by comparing Prunus and Pyrus (Malus). with the apple reference maps (‘Discovery’ and ‘Fiesta’), 66 Cipriani et al. (1999) found that only 18% of peach SSRs apple SSR loci could be positioned onto the homologous showed amplified bands in apple. Similarly, Yamamoto et LGs of pear (Yamamoto et al. 2007). Furthermore, SSR lo­ al. (2004a) observed that only 10% of the Prunus SSRs cus positions within LGs were almost identical in pear and could be transferred to the genetic linkage maps of Pyrus apple, indicating good co-linearity in all 17 LGs. Gisbert et (‘Bartlett’ and ‘Housui’). Only one out of 15 apple SSR al. (2009) used SSR markers from apple and pear to con­ markers was transferable to Prunus (Liebhard et al. 2002). struct genetic linkage maps of loquat cultivars ‘Algerie’ and A total of 613 RosCOS markers were successfully amplified ‘Zaozhong-6’; the loquat maps showed a high synteny with and mapped on the Prunus T × E reference map. These apple maps when anchored SSR markers were used. Fukuda RosCOS markers will be useful for further investigations of et al. (2014) identified almost perfect co-linearity of LG10 syntenic relationships between Pyrus (Malus) and Prunus. among loquat, pear, and apple. These findings suggest that Furthermore, several other reports have showed synteny all chromosomes of the genera in the tribe Pyreae show within Rosaceae plants (Sargent et al. 2009, Vilanova et al. co-linearity despite considerable differences in the genome 2008) and Rosaceae vs. other family (Staton et al. 2015). sizes, which range from 1.11 pg/2C to 1.57 pg/2C (Dickson et al. 1992, Dirlewanger et al. 2009b). Conclusion and perspectives

Co-linearity within Prunus In this manuscript, we describe to focus recent progress on The marker transferability is extremely high within whole-genome sequences, genome-wide SNP and SSR Prunus. For example, among 277 Prunus SSRs, including markers, construction of reference genetic linkage maps, 141 from peach (P. persica), 58 from apricot (P. armeniaca), and synteny studies in Rosaceae fruit trees, which will help 31 from almond (P. dulcis), 9 from sweet cherry (P. avium), us to develop new cultivars with desirable traits by MAS 4 from sour cherry (P. cerasus), and 6 from Myrobalan and new genomic-based strategies in breeding programs. plum ( Ehrh.), 95.3% showed PCR ampli­ Genetic improvement of Rosaceae fruit trees is strongly fication in Myrobalan plum (Dirlewanger et al. 2004a). hampered by their large tree size, long generation, an ex­ Furthermore, Mnejja et al. (2010) examined Prunus SSR tended juvenile phase for seedling (Luby and Shaw 2001, markers for transferability across rosaceous crops using Rikkerink et al. 2007). Therefore, it is considered that MAS nine species, almond (P. dulcis), peach (P. persica), apricot and marker-assisted breeding can accelerate selection and (P. armeniaca), Japanese plum ( Lindl.), reduce the progeny size and the cost of raising individuals European plum ( L.), sweet cherry to maturity in the field (Luby and Shaw 2001, Rikkerink et (P. avium), apple (M. × domestica), pear (P. communis), and al. 2007). However, attempts to MAS in fruit tree breeding strawberry (F. × ananassa). Of the 145 SSRs derived from programs remain limited for a few simply inherited traits, Prunus species, 83.6% of amplified bands of the expected because marker development for MAS via bi-parental QTL size range were identified in other Prunus species, and the mapping is also hindered by the same complications. Newly

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